R E S E A R C H A R T I C L E
A monocyte-TNF-endothelial activation axis in sickle transgenic
mice: Therapeutic benefit from TNF blockade
Anna Solovey
1|
Arif Somani
2|
John D. Belcher
1|
Liming Milbauer
1|
Lucile Vincent
1|
Rafal Pawlinski
3|
Karl A. Nath
4|
Robert J. Kelm Jr
5|
Nigel Mackman
3|
M. Gerard O
’
Sullivan
6|
Kalpna Gupta
1|
Gregory M. Vercellotti
1|
Robert P. Hebbel
11
Division of Hematology-Oncology-Transplantation, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota;2
Division of Critical Care, Department of Pediatrics, University of Minnesota Medical School;3
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; 4
Department of Medicine, Mayo Clinic, Rochester, Minnesota;5Department of Medicine, University of Vermont College of Medicine, Colchester, Vermont;6Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Minneapolis, Minnesota
Correspondence
Robert P. Hebbel, MD, MMC 480, 420 Delaware St. SE, Minneapolis MN 55455. Email [email protected]
Funding information
National Institutes of Health, Grant/Award Number: HL55552 to RPH; HL114567 to GV; and HL103773 to KG
Abstract
Elaboration of tumor necrosis factor (TNF) is a very early event in development of
ischemia/reperfu-sion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and
vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S1SAntilles, and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally
activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both
vessel wall and blood cells, and upon NFjB(p50) in a blood cell only. Involvement of TNF was
identi-fied by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an
IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the
murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count
and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion),
tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue
fac-tor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary
hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular
mean systolic pressure). In aggregate, these studies identify a prominent—and possibly dominant—
role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence,
plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should
be considered for human sickle cell anemia, a challenging but achievable translational research goal.
1
|I N T R O D U C T I O N
A chronic and robust systemic inflammatory state is a striking feature
and pathogenic factor in sickle cell anemia (SCA).1Hence, identification
of the core vector(s) underlying inflammation’s evolution and
perpetua-tion should identify useful therapeutic targets. As general underlying
processes, attention has focused upon vascular occlusion as the
initia-tor of ischemia/reperfusion injury (I/R) pathophysiology2 and upon
hemolysis as a source of toxic heme.3,4Beyond this, however, the role
of specific mediators as antecedent agents remains opaque in its
intricacy. Indeed, available data on SCA do not even enable parsing
potential mediators into those acting proximately versus more distally.
The literature on SCA, however, does document abnormal
activa-tion of blood monocytes and their ability to activate and/or damage
vascular endothelial cells in vitro.5–17This suggests monocyte
promi-nence in clinical disease genesis, if only because
monocyte/macro-phages are dominant generators of pro-inflammatory cytokines in the
broad context of inflammation’s generative role in vascular disease
generally.18 The present studies implicate a disease causing vector
extending from peripheral blood monocytes (PBM) to the vascular
endothelium, with the bridging mediator being tumor necrosis factor
(TNF, aka TNFa). Anna Solovey and Arif Somani contributed equally to this work.
...
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.VC2017 The Authors American Journal of Hematology Published by Wiley Periodicals, Inc.
Am J Hematol. 2017;92:1119–1130. wileyonlinelibrary.com/journal/ajh
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Our focus upon TNF stems from its roles as a“sentinel cytokine,”
largely post-transcriptionally regulated, and as an acute-phase initiator
of oxidant species and NFjB-driven (and other) responses that have
defensive, beneficial roles.19Yet, its obverse, maladaptive potential can
be realized when TNF is produced in excess and/or absent appropriate
resolution. Then, its pleiotropic effects can induce multi-faceted
inflam-matory pathology. Thus, it is the striking clinical benefit of TNF
block-ade in rheumatoid arthritis and other chronic inflammatory
diseases19,20that prompts our interest in this approach to the stubborn
chronicity of the SCA inflammatory state. Yet, the perplexing
complex-ity of TNF biology makes efficacy of TNF-blocking agents impossible
to predict with assurance.
This deserves exploration because many well-known TNF effects
are directly relevant to pathobiology of clinical sickle disease. To
illus-trate, we simply focus upon the vascular endothelium, the blood/tissue
interface of enormous importance in multiple biological processes.
Most globally harmful, TNF causes degradation of the glycocalyx,21
thus jeopardizing its critical roles that include: mediation of
shear-dependent functions (e.g., NO production); anchorage of surface
enzymes; and repelling potentially adherent blood cells. Separately,
TNF jeopardizes NO bioavailability by activating both endothelial
argi-nase (starving eNOS of its required substrate, arginine22) and
endothe-lial NADPH oxidase (depleting tetrahydrobiopterin to provoke
superoxide generation by eNOS23). Experimentally, TNF induces
endo-thelial adhesion molecule expression to promote RBC adhesion24and
vasoocclusion.25TNF exerts many additional adverse effects upon and
beyond endothelial cells.
At the level of clinical disease, TNF plays a prominent causative
role in organ diseases of general medicine, and these may be
instruc-tive regarding their counterparts in SCA. Examples include TNF’s role
in: pulmonary hypertension;26asthma;27sleep apnea;28left ventricular
dysfunction;29 cognitive, neuropsychiatric and neurologic
impair-ments;30and pain syndromes.31,32For most of these organ
manifesta-tions within general medicine, TNF blockade using etanercept has
yielded clinical improvement.
Therefore, TNF is a therapeutic target that should be considered
in SCA. The studies reported here examined effects of the TNF
blocker, etanercept, utilizing three sickle transgenic mouse models
that exhibit a systemic inflammatory state mimicking that of human
sickle disease.33,34 The resulting data create a framework within
which this intervention can be envisioned in the sickle disease
context.
Note that, due to complexity and variety of experiments,
interpre-tation of individual experiment sets is included in Results section, so
that Discussion can address the broader issues. (The data reported
here were presented, in preliminary form, at meetings of the American
Society of Hematology, 2007–2013).
2
|M A T E R I A L S / M E T H O D S
Some Methods are presented in greater detail in Supporting
Informa-tion Methods.
2.1
|Drugs
Etanercept,a chimeric fusion of human IgG1Fc domain and the 75 kDa
extracellular portion of human TNFR2,35acts as a“decoy”by binding
TNF. It is known to block TNF in murine experimental inflammatory
disease.36It also binds the lymphotoxin family, less understood
media-tors that use the same recepmedia-tors and mimic TNF itself. Our etanercept
dosing (3–10 mg/kg) falls in the lower end of the range used during its
preclinical development. We estimate that our highest dose would
pro-vide103-fold molar excess over murine blood TNF level.37By
com-parison, clinical trials of etanercept for human rheumatoid arthritis
used dosing that could have achieved a105-fold excess over human
TNF level.
Infliximabis a chimeric, mouse/human hybrid monoclonal specific
for TNF. Dosing here (10 mg/kg) would achieve the same fold-excess
as for our highest-dose etanercept. The typical human dose is 3 mg/kg,
given IV, every few weeks.
Anakinra, a recombinant form of the human IL-1 receptor
antago-nist, blocks signaling by IL-1aand IL-1b. We examined it because of prior use of an (undefined) IL-1 blocker by Kaul et al.38Dosing here
was 10 mg/kg, with unknown ratio to IL-1 receptor. Anakinra is known
to impede experimental murine inflammation.39
Scrambled Control Peptide, obtained via custom synthesis, contains
the 12 amino acids of the terminal end of the TNFR2 in scrambled order.
For simplicity and to economize on animal consumption, this was always
given at 0.1 mg/kg per dose, on same schedule as the other agents.
2.2
|Mice
Mice were housed and bred in our institution’s specific-pathogen-free
facility, with routine surveillance testing, to avoid confounding
infec-tious disease. Studies were done with the approval of our Institutional
Animal Care and Use Committee. We used NY1DD, S1SAntillesand SS-BERK sickle transgenic mice exhibiting, respectively, mild to moderate
to severe phenotypes.34 For NY1DD and S1SAntilles mice we used C57BL/6 controls. For mixed background BERK mice, our breeding
strategy ensured background homogenization between SS-BERK and
their AA-BERK controls.
2.2.1
|Cross-breeding
We separately crossbred knockout states for NFjB(p50)14and Egr-140
into NY1DD mice, using the same strategy we previously described.14
Additionally, we crossbred the NFjB(p50) knockout into SAntillesmice.
Then NFjB(p50)-/- NY1DD and NFjB(p50)-/- SAntilleswere crossbred
to ultimately obtain NFjB(p50)-/- S1SAntilles mice. Such knockout transfers always included>10 backcrosses against wild-type C57BL/6.
2.2.2
|Marrow transplantation
As described,14we previously conducted reciprocal marrow
transplanta-tions between wild-type and NFjB(p50)2/2NY1DD to obtain NY1DD mice having NFjB(p50)2/2in blood cells but not endothelium/tissue, or conversely having NFjB(p50)2/2 in endothelium/tissue but not blood cells. Using the same strategy, we here created NY1DD mice that
were Egr-1-/- in blood cells but not endothelium/tissue, or vice versa.
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2.3
|Study protocols
We tested the impact of drugs, transcription factor knockouts, or
trans-fusion of PBMC using animals at 3.5–4 months of age, unless
other-wise indicated. Experiments used pooled animals from multiple litters,
with pups from any one litter divided into both test and control groups,
using both males and females. Drug treatment studies always included
parallel controls receiving scrambled peptide (on same schedule as the
test drugs). Drugs were given by intra-peritoneal (ip) injection for
short-term experiments and by sub-cutaneous (sq) injection for
long-term experiments.
2.3.1
|Ischemia/reperfusion (I/R) model
Some NY1DD mice were subjected to hypoxia/reoxygenation (H/R)
stress: 3 hr at normobaric 8% O2, followed by reoxygenation (return to
room air, typically overnight).34This rapidly triggers actual ischemia/
reperfusion (I/R) in sickle–but not normal–mice.2,14,34Some animals
were pretreated with etanercept or infliximab or anakinra, at 10 mg/kg.
2.3.2
|Transfusion of PBMC (peripheral blood mononuclear
cells)
Using cardiac blood from pooled donors to isolate PBMC, we transfused
43106PBMC into naive recipient mice. Some PBMC preparations were monocyte depleted.14Some PBMC donor mice were post-H/R; some
donors or recipient mice were pretreated with etanercept at 10 mg/kg.
2.3.3
|Long-term therapeutic studies
We treated S1SAntilleswith etanercept (3 mg/kg, sq, once weekly, for 13 weeks), while BERK mice received higher dosing (3 mg/kg for 3
weeks or 6 mg/kg for 6 weeks, sq, twice weekly).
2.4
|Endpoints examined
2.4.1
|Blood counts and inflammatory markers
We performed CBC on cardiac blood using an analyzer standardized
for murine blood. We used ELISA kits to assess plasma biomarker
lev-els, and FACS to detect monocyte activation.
2.4.2
|Endothelial cell activation
We monitored expression of Tissue Factor (TF; expressed as the
per-cent of pulmonary veins positive) and vascular cell adhesion molecule 1
(VCAM-1; expressed as percent of 50610lm microvessels having >50% circumferential positivity).34
2.4.3
|Vascular stasis
Using S1SAntilles mice with implanted dorsal skinfold chambers, we determined the percentage of previously-flowing microvessels that
developed vascular stasis after provocation with either H/R or infusion
of hemin.3Some were pretreated with etanercept.
2.4.4
|Dermal Cytokines/neuropeptides
We tested cytokine/neuropeptide levels in conditioned medium from
skin biopsies (obtained from long-term etanercept-treated SS-BERK
mice) that were incubated ex vivo for 24 hours, as described.41
2.4.5
|Peri-vascular inflammatory aggregates
We used antibodies to CD11b and von Willebrand Factor (vWF) to
identify CD11b1 cells that were perivascular. We discovered this abnormality only retrospectively, so retrievable data are from frozen
lung sections that were not prepared with prospective intent to
quan-tify lesion prevalence.
2.4.6
|Pulmonary arteriolar muscularization
The same caveat applies here as well. We used antibodies to murine
smooth muscle actin (SMA) and vWF to score 50610lm microvessels as non-muscular, partially muscular, or fully muscular. A single value for each
mouse was derived from at least 50 vessels from three separated sections.
2.4.7
|Measurement of RVSP (right ventricular mean
systolic pressure)
Using isoflurane anesthesia, standardized mechanical ventilation, and a
para-sternal lateral thoracotomy,42we placed a right ventricular
pressure-transducing catheter to capture waveform data that enabled calculation
of RVSP. We measured multiple parameters to assess RV mass.
2.5
|Statistics
We made data comparisons using t testing, paired or unpaired, ANOVA
as indicated.
3
|R E S U L T S
We studied transgenic sickle mouse models having phenotypes ranging
from mild to severe: NY1DD, S1SAntillesand SS-BERK.34We identified endothelial activation by monitoring endothelial expression level of
tis-sue factor (TF) and VCAM-1. Studies utilized TNF blocker etanercept
(E), with a control peptide (CP) always examined in parallel; the latter
uniformly failed to exert any effect compared to no treatment.
There-fore, to simplify graphics, we focus upon only the most important
com-parisons. An interpretation of individual experiment sets is included
within Results, so Discussion can address the broader issues.
4
|I D E N T I F I C A T I O N O F A M O N O C Y T E
-T N F - E N D O -T H E L I A L A C -T I V A -T I O N A X I S
4.1
|Activated monocytes cause endothelial
activation in sickle mice (Figure 1)
Using all three sickle mouse models, we demonstrated the importance
of peripheral blood monocytes (PBM) via transfusion experiments in
which peripheral blood mononuclear cells (PBMC) were infused into
appropriate control recipient mice.
To illustrate context, our relevant previously published
experi-ment14is illustrated here as Figure 1A. When infused into naive (i.e.,
unstressed) NY1DD recipients, the PBMC harvested from control
NY1DD did not activate endothelial TF (bar 1); but infusion of PBMC
from H/R-stressed NY1DD donors did do so (bar 2). This activating
depleted of PBM (bar 3). Thus, PBM activation accounts for the
endo-thelial activation state that is triggered when NY1DD mice are
experi-mentally stressed with H/R exposure to induce I/R.2,14,34Therefore, we
here sought to corroborate this using sickle mice that spontaneously
exhibit an inflammatory state, i.e., not requiring challenge by a stressor.
4.1.1
|S
1
S
Antillesmice
Infusion of PBMC from S1SAntillesdonors (Figure 1B, bar 2), but not infusion of PBMC from C57BL6 control donors (Figure 1B, bar 1),
acti-vated TF in C57BL6 recipient mice. This activating effect was lost if
the S1SAntillesdonors concurrently were NFjB(p50)-/- (Figure 1B, bar 3). This is supportive because we previously had demonstrated, using
NY1DD mice, that NFjB(p50)-/- eliminates ability of PBMC to activate
endothelium.14
4.1.2
|SS-BERK mice
Using BERK mice, we also identified the activating role of PBMC on
endothelial TF (Figure 1C), as well as endothelial VCAM-1 (Figure 1D).
In AA-BERK recipients, activation of both endothelial antigens resulted
from infusion of PBMC from SS-BERK donors (bars 2) but not from
AA-BERK control donors (bars 1).
4.2
|Endothelial activation is TNF-dependent in sickle
mice
We identified involvement of TNF first using the H/R stressed NY1DD
mice that developed increased expression of both TF and VCAM-1
(Figure 2A,B, bars 2), compared to baseline expression in unstressed
control NY1DD (bars 1). This activation was unaffected by
F I G U R E 1 Transfusion experiments identify the role of PBM in endothelial cell activation. Peripheral blood mononuclear cells (PBMC) from donor mice were transfused into recipient mice, and pulmonary endothelial activation was monitored via expression of TF and/or VCAM-1. Some mice were pretreated either with etanercept (1E) or control peptide (1CP). Since the CP had no impact compared to wholly unmanipulated control animals, that is not shown.Panel A. Transfusion of NY1DD PBMC (from unstressed donors) into naive NY1DD recipients did not increase recipient endothelial TF (bar 1). PBMC from post-H/R NY1DD donors did do so (bar 2), an effect ame-liorated by specific depletion of PBM from the PBMC population (bar 3). For Panel A,n53,3,3. Previously reported,14this single experi-ment is reproduced in Panel A to establish context.Panel B.Transfusion of C57BL6 PBMC into C57BL6 recipients did not activate recipient TF (bar 1), but transfusion of S1SAntillesPBMC did do so (bar 2). This impact of S1SAntillesPBMC was eliminated if the donor mice had concurrent NFB(p50)-/- (bar 3) For Panel B,n55,6,6.Panel C.AA-BERK PBMC transfused into AA-BERK recipients did not activate TF expression (bar 1), but SS-BERK PBMC did do so (bar 2). Pretreatment of the PBMC donors with either control peptide (bar 2) or eta-nercept (bar 3) had no effect. However, pretreatment of the PBMC recipients with etaeta-nercept greatly blunted the TF activating effect (bar 5). For Panel C,n53,3,3,3,3.Panel D.For the same mice shown in Panel C, etanercept pretreatment of either PBMC donors (bar 3) or PBMC recipients (bar 5) reduced recipient VCAM-1 expression. For Panel D,n53,3,3,3,3
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pretreatment with control peptide (CP, bars 3) or heat-inactivated
eta-nercept (bars 4). However, pretreatment with intact etaeta-nercept clearly
exerted dose-dependent inhibition (bars 5 and 6), also seen using the
TNF-specific inhibitor, infliximab (bars 8). By comparison, the IL-1
receptor inhibitor, anakinra, was a less potent inhibitor of TF than was
etanercept; but for VCAM-1 inhibition, anakinra exhibited potency
equivalent to etanercept (bars 7).
4.3
|Relationship between PBM and TNF in
endothelial activation
Because the biology of TNF is so complex (see Discussion), we
exam-ined this apparent PBM-TNF link in more detail, using PBMC
transfu-sions and the BERK model. AA-BERK recipient mice that received
PBMC from AA-BERK donors exhibited the lower baseline expression
of TF (Figure 1C) and VCAM-1 (Figure 1D) (bars 1). But recipients that
received PBMC from SS-BERK donors developed activation with
higher expression of both TF and VCAM-1 (bars 2). These starting data
enable interpretation of the following experiment.
4.3.1
|TF
When we pretreated the SS-BERK donors of with etanercept, their
PBMC still induced endothelial TF expression in the AA-BERK
recipi-ents (Figure 1C, bar 3). However, when we pretreated the AA-BERK
recipients with etanercept, the subsequently transfused SS-BERK
PBMC were unable to activate endothelial TF in those recipients (bar
5). Thus, etanercept’s blockade of endothelial TF expression apparently
requires the drug to be present when the active PBMC are present. In
turn, this implies that etanercept acts either by binding sTNF in plasma
or by engaging with tmTNF on endothelial cells themselves.
4.3.2
|VCAM-1
When we examined the same mice for endothelial VCAM-1
expres-sion, a different result emerged. The elevation of recipient VCAM-1
caused by transfusion of SS-BERK PBMC (Figure 1D, bar 2) was
ame-liorated by etanercept pretreatment of either the PBMC donor (bar 3)
or the PBMC recipient (bar 5). Thus, etanercept’s blockade of
endothe-lial VCAM-1 expression presumably results from the drug modifying
the PBMC themselves. One possibility is that etanercept could engage
tmTNF on the PBM, with a consequent down-regulating effect on their
ability to produce TNF, an effect that persisted when the PBM were
then transfused.
4.4
|Divergence of TF versus VCAM-1 activating
mechanisms
Thus, results described so far include two suggestions that activation
of endothelial TF and VCAM-1 expression in the sickle I/R context
may involve somewhat differing mechanisms. First, the results of
treat-ing mice with the TNF blocker etanercept vs. the IL-1 blocker anakinra
suggest that endothelial TF and VCAM-1 may have non-identical
underlying mechanisms (Figure 2A,B). Second, the data probing the
PBM/etanercept relationship suggested that etanercept may be
targeting different features of TNF biology in its inhibition of TF vs
VCAM-1 (Figure 1C,D). For this reason, we here describe ancillary
experiments that bear on mechanistic divergence of etanercept action
and its potential implications for therapeutics.
We focused upon NFjB and Egr-1 because these are dominant
regulators of TF expression.
4.4.1
|Egr-1
Using the post-H/R NY1DD model, we observed that Egr-1-/-
elimi-nated the increased endothelial TF expression induced by sickle I/R.
This was true both if Egr-1-/- was in endothelium/tissue but not blood
cells, and if Egr-1-/- was in blood cells but no endothelium/tissue
(Sup-porting Information Figure S1). These findings are consistent,
respec-tively, with Egr-10s known prominence as a regulator of TF expression43 and with its suspected role in monocyte TNF
expression.44
4.4.2
|NF
j
B(p50)
We previously reported that the impact of PBM on endothelial TF
expression actually requires a NFjB(p50) dependent gene within blood
cells.14It is relevant that NF
jB(p50) is believed to participate indirectly in PBM production of TNF.45 Interestingly, however, we have now
observed that NFjB(p50)-/- in sickle mice actually causes increased
expression of endothelial VCAM-1 (Supporting Information Figure S1).
This can be explained simply by the NFjB p50/p65 heterodimer’s less
robust promotion of gene expression, compared to that of the NFjB
p65/p65 homodimer.46Hence, a knockout of p50 effectively removes
a braking effect.
4.4.3
|NF
j
B inhibitors
These data suggest it may be advisable to avoid NFjB inhibitors that
are truly specific for the p50 component. Fortunately, most are not
p50 specific. Nonetheless, we here examined the VCAM-1 expression
impact of the NFjB inhibitors we previously identified as effective
inhibitors of endothelial TF in sickle mice.14,34,47 We find several of
those agents to be effective also in ameliorating the increased
VCAM-1 in post-H/R NYVCAM-1DD mice: lovastatin, andrographolide, curcumin,
sul-fasalazine and histone deacetylase inhibitors (Supporting Information
Table S1).
This VCAM-1 expression scoring was enabled by our discovery,
during the course of these studies of an unsuspected, regional
hetero-geneity in the dynamism of VCAM-10s activation in sickle mice. VCAM-1 responses, both increases and inhibitions, were detectable
only by focusing on vessels 50610lm diameter.
5
|T H E R A P E U T I C B E N E F I T F R O M T N F
B L O C K A D E
To assess etanercept in a therapeutic context, we conducted long-term
studies giving etanercept vs. control peptide toS1SAntillesor SS-BERK mice. Doses, schedules and length of treatment are described in
Meth-ods. Etanercept exerted a beneficial impact upon nearly all of the
F I G U R E 2 TNF blockade ameliorates endothelial activation in sickle mice.Panel A. The low TF expression of unstressed NY1DD mice (bar 1) is increased by H/R exposure (bar 2). Pretreatment with control peptide (bar 3) or with heat inactivated etanercept (bar 4) had no effect, but etanercept exerted dose-dependent inhibition (bar 5 and 6). IL-1 blocker anakinra had very little effect (bar 7), but TNF-specific blocker infliximab strongly inhibited TF (bar 8). For Panel A,n58,8,8,4,10,3,8,5.Panel Bshows inhibition of VCAM-1 expression in those same NY1DD mice. For Panel B,n510,10,8,3,8,3,5,4.Panels C and D. S1SAntillesmice had elevated TF and VCAM-1 (bars 2), compared to their C57BL controls (bars 1). After long-term treatment (13 weeks), this was inhibited by etanercept (bars 4) but not control peptide (bars 3). For Panel C,n57,4,6,11. For Panel D,n57,4,5,6.Panels E and F. SS-BERK mice had elevated TF and VCAM-1 (bars 2), compared to their AA-BERK controls controls (bars 1). Etanercept treatment for 3 weeks (bars 4) or 6 weeks (bars 6) inhibited TF and VCAM-1 expression; control peptide did not (bars 3 and 5). For Panels E and F,n56,4,5,6,3,4
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5.1
|Inflammatory biomarkers (Table 1)
In general, blood biomarkers of inflammation responded favorably to
etanercept, with significant improvements in: soluble VCAM-1
(sVCAM-1), a marker of endothelial activation; SAP (serum amyloid
P), the murine equivalent of human CRP; TNF itself; and MCP-1
(monocyte chemotactic protein 1) elaborated, e.g., by endothelial
cells in response to TNF. Serum neopterin, a product of monocyte
activation, was unhelpful. Blood monocyte activation status (by
FACS) revealed a response to etanercept in the proportion of
mono-cytes labeling positively for TNF; but proportions of CD11b1or IL-61did not respond.
Long-term treatment of S1SAntilles mice with etanercept also improved blood Hb and platelet count. An abnormally low platelet
count is a feature of murine sickle models (Supporting Information
Table S2), as it is in some humans with sickle cell anemia.
5.2
|Pulmonary endothelial activation state
Etanercept was an effective inhibitor of abnormal pulmonary
endo-thelial cell expression of TF and VCAM-1 in all three sickle mouse
models models (Figure 2): the acute I/R triggered in NY1DD by H/R
exposure (panels A and B); and the spontaneous, chronic I/R state
of S1SAntilles(panels C and D) and SS-BERK (panels E and F). Nota-bly, the SS-BERK mice revealed that the suppression of TF
expres-sion increased further with longer duration of etanercept
treatment.
5.3
|Neuro-inflammatory mediators and pain
In vitro incubation of skin biopsies from SS-BERK mice that had been
treated long-term with control peptide or etanercept enabled
quantita-tive measurement of neuro-inflammatory mediator elaboration from
actual tissue. As previously reported, such data actually correlate with
pain behaviors exhibited by sickle mice.41 Biopsies from the
etanercept-treated mice exhibited decreased elaboration of IL-6,
MCP-1, substance P and CGRP (calcitonin gene-related peptide) (Supporting
Information Figure S2). Although not achieving significance due to
small number of animals available, levels of IL-1b, IL-1a, IFNg, and MIP-1asuggested a downwards trend.
We know that the biologies of acute and chronic pain both involve
inflammation having complicated, bidirectional effects between
sys-temic and neuro-inflammatory substances.31,32 Pain behaviors could
not be tested here, as the number of available animals was far too
small. Yet, the present data suggest that therapy with etanercept might
benefit pain.
5.4
|Histopathology
In contrast to an absence of pathology in C57BL6 control mice, livers
of S1SAntillesmice displayed acute or chronic coagulative necrosis, indi-cating ischemia or prior infarction. This was ameliorated both by
long-term treatment with etanercept and, revealingly, by presence of the
NFjB(p50)-/- state (Supporting Information Table S2).
5.5
|Vascular occlusion
To assess etanercept impact upon acute vascular flow deficiency, we
studied S1SAntillesmice bearing dermal windows (to enable microvas-cular flow visualization). We pretreated them with etanercept or
con-trol peptide and then challenged them with an insult known to trigger
vascular stasis, either H/R2,14,34or infusion of hemin.3The stasis
trig-gered by H/R was greatly diminished by etanercept (Figure 3A), but
that triggered by hemin infusion was not blunted significantly (Figure
3B). This is consistent with our current understanding of these two
sta-sis induction mechanisms. H/R exposure triggers I/R14,34 which
involves TNF elaboration in its very earliest stages. In contrast, hemin
directly perturbs endothelium, causing stasis via TNF-independent
mechanisms in addition to TNF elaboration.3
5.6
|Pulmonary arterial disease
We examined three surrogate markers for presence of pulmonary
hypertension.
5.6.1
|Peri-vascular inflammatory aggregates
Staining for CD11b1evinced the presence of perivascular inflammatory cell aggregates (examples shown in Supporting Information Figure S3).
T A B L E 1 Effects of long-term TNF blockade in S1S sickle micea
Long-term Treatment Agent
Parameters CP E P.
Blood counts
Hb (g/dl) 9.111.3 (10) 11.261.1 (9) .032 Reticulocytes (%) 4.161.1 (5) 3.562.5 (4) .674 WBC (3103/lL) 9.066.8 (9) 7.365.3 (9) .564 Platelets (3103/lL) 8346125 (6) 1249646 (4) .0006 Biomarkers
sVCAM-1 (ng/mL) 1053684 (6) 827655 (5) .027 SAP (lg/mL) 5556101 (5) 3676115 (5) .025 TNF (pg/mL) 14.766.1 (4) 3.560.16 (5) .0053 MCP-1 (pg/mL) 60.9629.0 (4) 23.2613.9 (5) .0359 Neopterin (pg/mL) 650624 (6) 6006165 (5) ns Monocyte Activation
TNF1 (%) 13.261.0 (12) 4.663.3 (12) .001 CD11b1 (MFI) 257613 (12) 229621 (12) ns IL-61 (MFI) 20.163.6 (12) 25.0614.8 (12) ns a
Since the available material allowed only semi-quantitation, we
applied three different measures (Table 2). Each parameter revealed
increased prevalence of these inflammatory aggregates in S1SAntilles compared to C57Bl6 control, and each was notably diminished in
response to etanercept. Because such perivascular inflammatory
aggregates are part of the histopathology of human pulmonary
hyper-tension,48this result prompted the following additional studies.
5.6.2
|Pulmonary arteriolar muscularization
Staining for smooth muscle actin (SMA) (examples shown in Supporting
Information Figure S3) suggested a trend towards increasing
musculari-zation of50lm pulmonary arterial vessels as animals matured from
age 1 to 4 months, seen for both C57BL6 and S1SAntillesmice (Figure 4A). However, at both ages, muscularization was greater for sickle than
for normal mice. Notably, after 3 months of etanercept therapy
(start-ing at age 1 month) the muscularization in S1SAntillesmice was signifi-cantly reduced. Indeed, it appears the etanercept may have both
prevented further muscularization and somewhat reduced existing
muscularization.
5.6.3
|Elevated right ventricular pressure
As a surrogate measure for possible elevation of pulmonary mean
arte-rial pressure (i.e., pulmonary hypertension), we found that right
ventric-ular (mean) systolic pressure (RVSP) was elevated in S1SAntillesmice compared to C57Bl6 controls. Treatment with etanercept for 6 weeks
(started at weaning) eliminated RVSP elevation in sickle mice (Figure
4B). After 13 weeks of therapy, however, the differential between
groups was preserved, but the statistical significance was lost. This loss
may simply be because, as inspection indicates (Figure 4B),
inter-individual variability increased markedly as age advanced (as often
happens in clinical complications of human SCA). The small number of
animals precluded resolution of this.
For both treatment durations, the accompanying necropsy
meas-ures of right heart weights (RV/RV1LV; RV/tibia length; RV/body weight) did not reveal a significant change in response to etanercept in
this study.
6
|D I S C U S S I O N
The present studies identify a monocyte-TNF-endothelial activation
axis promotive of pleiotropic disturbances and damage. Results were
consistent for the sickle mice of two different genetic backgrounds,
F I G U R E 3 TNF blockade eliminates H/R-triggered vascular stasis. S1SAntillesmice were pretreated either with control peptide (CP) or etanercept (E) and then provoked with H/R exposure or with infusion of hemin; stasis of previously-flowing vessels was scored after 1 and 4 hours.Panel A. Pretreatment with etanercept eliminated H/R-triggered stasis, its normal level in absence of H/R stress being<10%. Number of animals: 11,10,11,10.Panel B. Etanercept failed to significantly blunt stasis triggered by hemin infusion. Number of animals: 6,6,6,6
T A B L E 2 Prevalence of peri-vascular inflammatory aggregates (CD11b1cells)a
C57BL6 S1Santilles Semi-quantification
method . control unRx CP E .
mice with large aggregates (n)
0/5 4/6 4/6 0/11
vessels with smaller aggregate (n)b
968 4769 4566 22618
cells per vessel (n)c 1.560.3 3.660.4 2.960.2 2.260.5 a
S1SAntillesmice were untreated (unRx) or treated either with control peptide (CP) or etanercept (E) for 13 weeks. Data for small aggregates and cells/vessel are expressed as mean6SD. Definitions: large and small aggregates were>20 cells or 3–20 cells, respectively, around any given vessel. Above 20, they were too numerous to be discretely identified or counted.
b
Untreated S1SAntillesvs. C57,P5.00034. CP vs. E treated S1SAntilles mice,P5.0038.
cUntreated S1SAntillesvs. C57,P52.431025. CP vs. E treated S1SAntilles,P5.042.
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and they were similar for both experimentally triggered I/R (NY1DD
mice) and mice having chronic spontaneous I/R (S1SAntilles and SS-BERK mice). We identified a substantial ameliorating effect of the TNF
blocker, etanercept, upon nearly all endpoints evaluated, a diverse
group of clinical features shared by sickle mice and humans. Thus,
interruption of this monocyte-TNF-endothelial vector offers an
inter-esting therapeutic targeting option.
6.1
|Interpreting results of TNF blockade
The complexity of TNF biology renders interpretation of experiments
challenging.19,20Signaling derives from both soluble TNF (sTNF) and
cell-bound trans-membrane TNF (tmTNF). The sTNF activates via both
receptors, TNFR1 and TNFR2; mTNF activates only via latter. One
cell’s tmTNF can receive outside-in activating signals if it engages
another cell’s TNFR. Conversely, a TNF blocking antibody may induce
positive or negative signaling within a cell upon engaging its tmTNF.49
Finally, plasma contains released TNFRs that can bind TNF. Despite
this bewildering intricacy, its net impact and TNF-dependence are
experimentally demonstrable. In the present studies, the impact of
eta-nercept was uniformly beneficial.
6.2
|Evidence for a TNF role in sickle cell anemia
The SCA inflammatory state guarantees participation of TNF, the
ques-tion being with what primacy. sTNF levels often are elevated in
SCA,50,51 but this itself may not be very useful information, as has
been discovered in the classical TNF-dependent disease, rheumatoid
arthritis (RA). Nonetheless, the very spectrum of clinical complications
in SCA is consistent with TNF being a participating inflammatory
insti-gator (per Introduction).
Actually, RA provides an instructive paradigmatic context. Since
humans having the TNF(-308) promoter GG polymorphism are
predis-posed to develop inflammatory RA,52it is interesting that SCA children
having that G allele are predisposed to develop large vessel stroke53
that is caused by an inflammatory vasculopathy in the Circle of Willis.1
Indeed, we found that endothelial cells from sickle children with Circle
of Willis disease exhibited an exaggerated NFjB activation response to
stimulation with TNF/IL-1.54
Thus, current concepts about TNF and inflammation generally, and
disease consequent to the sickle mutation specifically, are consistent
with the conclusions we draw from the present studies.
6.3
|Is TNF actually the most proximate mediator?
This really is the wrong question. Inflammatory signaling, rather than a
strictly hierarchical cascade, is a vast and intricate network. To
illus-trate, Kaul et al. preliminarily reported that blockade of IL-1b amelio-rated endothelial activation and inflammation in a sickle mouse model
(probing only VCAM-1).38This is perfectly consistent with our present
observations. Moreover, TNF and IL-1bexert overlapping effects, and each induces production of the other. Thus, our results reveal only that
the participation of TNF is substantial enough that its inhibition is
unambiguously effective and beneficial.
Our results do beg the question: How are sickle monocytes
acti-vated in the first place? This is not yet answerable, as many mediators
within the sickle milieu are capable of doing so; it seems likely that
mul-tiple agents are activating. Nonetheless, we find three TLR4 ligands to
be highly likely actors in SCA: free heme from hemolysis, HMGB1 (high
mobility group box 1) released during I/R, and heparan sulfate released
from endothelial glycocalyx. Each of these ligands can be expected to
F I G U R E 4 TNF blockade benefits surrogate measures of pulmonary hypertension.Panel A. Pulmonary arteriolar muscularization in S1SAntilles mice is plotted, each bar showing percent of arterioles with full (black), partial (hatched), or no (white) muscularization, defined by staining for SMC actin. [Examples of SMC actin staining images are provided in Supporting Information Figure S3]. Mouse age is shown in months. TheP
exert activating effects via TLR4 on monocytes, leading to TNF release;
and TNF actually enhances activity of this TLR4 pathway.55This would
seem to predict the very cyclicity and apparent perpetuity of
inflamma-tion that is evident in SCA.
6.4
|Why TNF blockade should work in sickle disease
The remarkably uniform efficacy of etanercept seen here is, we
sus-pect, not because it is exerting“anti-inflammatory”effects in the
tradi-tional sense. Rather, it is impeding the actual triggers that comprise the
veryinceptionof I/R pathobiology.2If so, TNF blockade would inhibit – perhaps even halt—the very driver of the perpetual inflammatory
pro-cess responsible for the vascular dysfunction of SCA. If this I/R model
of SCA pathophysiology2 is valid, TNF can be expected to be
ameliorative.
6.5
|What next?
The unavoidable uncertainty inherent in the complexity of TNF biology
also complicates therapeutic predictability. Beyond data such as ours,
only advancing to pilot study in humans can answer whether or not
etanercept may exert benefit for SCA. This, however, may require our
field to create solutions to several barriers, as we expect efficacy of
etanercept might be exerted over longer rather than shorter
time-scales. Additionally, nature and mechanism of benefit could be different
for acute intervention vs. chronic prevention.
Finally, we are not arguing that etanercept itself would be an ideal
therapeutic, because it is expensive and must be administered
subcuta-neously, although it could be deployed immediately in health systems
that can deal with such issues. As alternatives, ongoing development of
small molecule TNF blockers may eventually offer very wide
applicabil-ity and accessibilapplicabil-ity. Meanwhile, it would be helpful for us to glean
insights about pathophysiology that would derive from a focused pilot
test of etanercept in humans with SCA. Attempting that would present
several significant challenges for which solutions have been proposed
elsewhere. Indeed, that might identify additional opportunities for
pro-ductive therapeutic targeting.
A C K N O W L E D G M E N T S
We thank Jim Kiley, Fuad Abdullah, Sethu Nair, and Chunsheng
Chen for technical assistance. We are grateful for editorial expertise
provided by Tracy Daye-Groves and Michael Franklin. This work
was supported by the National Institutes of Health: HL55552 to
RPH; HL114567 to GV; and HL103773 to KG.
C O N F L I C T O F I N T E R E S T
The authors have no conflicts of interest.
A U T H O R C O N T R I B U T I O N S
Conceived, designed, supervised and guided study: AS, AS, KG, GV,
RPH. Conducted experiments: AS, AS, JB, LV, LM, KAN, GOS.
Provided critical models, reagents and expertise: RJK, RP, NM, RPH.
Wrote or edited manuscript: AS, AS, NM, GOS, JB, RPH.
R E F E R E N C E S
[1] Hebbel RP, Osarogiagbon R, Kaul D. The endothelial biology of sickle cell disease: inflammation and a chronic vasculopathy. Micro-circulation.2004;11(2):129–151.
[2] Hebbel RP. Ischemia-reperfusion injury in sickle cell anemia: rela-tionship to acute chest syndrome, endothelial dysfunction, arterial vasculopathy, and inflammatory pain.Hematol Oncol Clin North Am.
2014;28(2):181–198.
[3] Belcher JD, Chen C, Nguyen J, et al. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.Blood.2014;123(3):377–390.
[4] Setty BN, Betal SG, Zhang J, Stuart MJ. Heme induces endothelial tis-sue factor expression: potential role in hemostatic activation in patients with hemolytic anemia.J Thromb Haemost.2008;6(12):2202–2209.
[5] Belcher JD, Marker PH, Weber JP, Hebbel RP, Vercellotti GM. Acti-vated monocytes in sickle cell disease: potential role in the activa-tion of vascular endothelium and vaso-occlusion.Blood.2000;96(7): 2451–2459.
[6] Wun T, Cordoba M, Rangaswami A, Cheung AW, Paglieroni T. Acti-vated monocytes and platelet-monocyte aggregates in patients with sickle cell disease.Clin Lab Haematol.2002;24(2):81–88.
[7] Selvaraj SK, Giri RK, Perelman N, Johnson C, Malik P, Kalra VK. Mech-anism of monocyte activation and expression of proinflammatory cyto-chemokines by placenta growth factor.Blood.2003;102(4):1515–1524.
[8] Perelman N, Selvaraj SK, Batra S, et al. Placenta growth factor acti-vates monocytes and correlates with sickle cell disease severity.
Blood.2003;102(4):1506–1514.
[9] Sloma I, Zilber MT, Charron D, Girot R, Tamouza R, Gelin C. Upreg-ulation and atypical expression of the CD1 molecules on monocytes in sickle cell disease.Hum Immunol.2004;65(11):1370–1376.
[10] Zennadi R, Chien A, Xu K, Batchvarova M, Telen MJ. Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium.
Blood.2008;112(8):3474–3483.
[11] Marcal LE, Dias-da-Motta PM, Rehder J, et al. Up-regulation of
NADPH oxidase components and increased production of interferon-gamma by leukocytes from sickle cell disease patients.
Am J Hematol.2008;83(1):41–45.
[12] Brittain JE, Knoll CM, Ataga KI, Orringer EP, Parise LV. Fibronectin bridges monocytes and reticulocytes via integrin alpha4beta1.Br J Haematol.2008;141(6):872–881.
[13] Chaar V, Picot J, Renaud O, et al. Aggregation of mononuclear and red blood cells through an {alpha}4{beta}1-Lu/basal cell adhesion molecule interaction in sickle cell disease.Haematologica. 2010;95 (11):1841–1848.
[14] Kollander R, Solovey A, Milbauer LC, Abdulla F, Kelm RJ, Jr, Hebbel RP. Nuclear factor-kappa B (NFkappaB) component p50 in blood mononuclear cells regulates endothelial tissue factor expression in sickle transgenic mice: implications for the coagulopathy of sickle cell disease.Transl Res.2010;155(4):170–177.
[15] Safaya S, Steinberg MH, Klings ES. Monocytes from sickle cell dis-ease patients induce differential pulmonary endothelial gene expres-sion via activation of NF-kappaB signaling pathway. Mol Immunol.
2012;50(1–2):117–123.
[16] Setty BN, Key NS, Rao AK, et al. Tissue factor-positive monocytes in children with sickle cell disease: correlation with biomarkers of haemolysis.Br J Haematol.2012;157(3):370–380.
1128
|
A
J
H
[17] Ragab SM, Soliman MA. Tissue factor-positive monocytes expres-sion in children with sickle cell disease: clinical implication and rela-tion to inflammatory and Coagulation Markers. Blood Coagul Fibrinolysis.2016;27(8):862–869.
[18] Sprague AH, Khalil RA. Inflammatory cytokines in vascular dys-function and vascular disease.Biochem Pharmacol.2009;78(6):539– 552.
[19] Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP. Tumor necro-sis factor antagonist mechanisms of action: a comprehensive review.Pharmacol Ther.2008;117(2):244–279.
[20] Van Hauwermeiren F, Vandenbroucke RE, Libert C. Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1.Cytokine Growth Factor Rev.2011;22(5–6):311–319.
[21] Schmidt EP, Yang Y, Janssen WJ, et al. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.Nat Med.2012;18(8):1217–1223.
[22] Gao X, Xu X, Belmadani S, et al. TNF-alpha contributes to endothe-lial dysfunction by upregulating arginase in ischemia/reperfusion injury.Arterioscler Thromb Vasc Biol.2007;27(6):1269–1275.
[23] Crabtree MJ, Channon KM. Synthesis and recycling of tetrahydro-biopterin in endothelial function and vascular disease.Nitric Oxide.
2011;25(2):81–88.
[24] Swerlick RA, Eckman JR, Kumar A, Jeitler M, Wick TM. Alpha 4 beta 1-integrin expression on sickle reticulocytes: vascular cell adhesion molecule-1-dependent binding to endothelium. Blood.
1993;82(6):1891–1899.
[25] Kaul DK, Fabry ME, Nagel RL. Microvascular sites and characteris-tics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications.Proc Natl Acad Sci U S A.1989;86(9):3356–3360.
[26] Fujita M, Mason RJ, Cool C, Shannon JM, Hara N, Fagan KA. Pul-monary hypertension in TNF-alpha-overexpressing mice is associ-ated with decreased VEGF gene expression.J Appl Physiol ( Physiol).
2002;93(6):2162–2170.
[27] Berry M, Brightling C, Pavord I, Wardlaw A. TNF-alpha in asthma.
Curr Opin Pharmacol.2007;7(3):279–282.
[28] Vgontzas AN, Zoumakis E, Lin HM, Bixler EO, Trakada G, Chrousos GP. Marked decrease in sleepiness in patients with sleep apnea by etanercept, a tumor necrosis factor-alpha antagonist.J Clin Endocri-nol Metab.2004;89(9):4409–4413.
[29] Bozkurt B, Kribbs SB, Clubb FJ, Jr., et al. Pathophysiologically rele-vant concentrations of tumor necrosis factor-alpha promote pro-gressive left ventricular dysfunction and remodeling in rats.
Circulation.1998;97(14):1382–1391.
[30] Tobinick E, Kim NM, Reyzin G, Rodriguez-Romanacce H, DePuy V. Selective TNF inhibition for chronic stroke and traumatic brain injury: an observational study involving 629 consecutive patients treated with perispinal etanercept.CNS Drugs. 2012;26(12):1051– 1070.
[31] Marchand F, Perretti M, McMahon SB. Role of the immune system in chronic pain.Nat Rev Neurosci.2005;6(7):521–532.
[32] Hess A, Axmann R, Rech J, et al. Blockade of TNF-a rapidly inhibits pain responses in the central nervous system. Proceed-ings of the National Academy of Sciences. 2011;108(9):3731– 3736.
[33] Belcher JD, Bryant CJ, Nguyen J, et al. Transgenic sickle mice have vascular inflammation.Blood.2003;101(10):3953–3959.
[34] Solovey A, Kollander R, Shet A, et al. Endothelial cell expression of tissue factor in sickle mice is augmented by hypoxia/reoxygenation and inhibited by lovastatin.Blood.2004;104(3):840–846.
[35] Peppel K, Crawford D, Beutler B. A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.J Exp Med.1991;174(6):1483–1489.
[36] Mazzon E, Esposito E, Di Paola R, et al. Effect of tumour necrosis factor-alpha receptor 1 genetic deletion on carrageenan-induced acute inflammation: a comparison with etanercept. Clin Exp Immu-nol.2008;153(1):136–149.
[37] Luong BT, Chong BS, Lowder DM. Treatment options for rheuma-toid arthritis: celecoxib, leflunomide, etanercept, and infliximab.Ann Pharmacother.2000;34(6):743–760.
[38] Kaul DK, Thangaswamy S, Suzuka SM, Fabry ME, Alan A. Wanderer and Hermann Gram Anti-interleukin-1B antibody-based therapy ameliorates endothelial activation and inflammation in sickle mice.
Blood. 2001;118:848. [abstract]
[39] Toldo S, Schatz AM, Mezzaroma E, et al. Recombinant human interleukin-1 receptor antagonist provides cardioprotection during myocardial ischemia reperfusion in the mouse. Cardiovasc Drugs Ther.2012;26(3):273–276.
[40] Pawlinski R, Pedersen B, Kehrle B, et al. Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model.Blood.2003;101(10):3940–3947.
[41] Vincent L, Vang D, Nguyen J, et al. Mast cell activation contributes to sickle cell pathobiology and pain in mice. Blood. 2013;122(11): 1853–1862.
[42] Cruz JA, Bauer EM, Rodriguez AI, et al. Chronic hypoxia induces right heart failure in caveolin-1-/- mice. Am J Physiol Heart Circ Physiol.2012;302(12):H2518–H2527.
[43] Mackman N. Regulation of the tissue factor gene.FASEB J.1995;9 (10):883–889.
[44] Pritchard MT, Nagy LE. Ethanol-induced liver injury: potential roles for egr-1. Alcohol Clin Exp Res. 2005;29(11 Suppl):146S– 150S.
[45] Joyce DA, Gimblett G, Steer JH. Targets of glucocorticoid action on TNF-alpha release by macrophages. Inflamm Res. 2001;50(7):337– 340.
[46] Parry GC, Mackman N. A set of inducible genes expressed by acti-vated human monocytic and endothelial cells contain kappa B-like sites that specifically bind c-Rel-p65 heterodimers. J Biol Chem.
1994;269(33):20823–20825.
[47] Hebbel RP, Vercellotti GM, Pace BS, et al. The HDAC inhibitors tri-chostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle trans-genic mice.Blood.2010;115(12):2483–2490.
[48] Robinson JC, Graham BB, Rouault TC, Tuder RM. The crossroads of iron with hypoxia and cellular metabolism. Implications in the path-obiology of pulmonary hypertension. Am J Respir Cell Mol Biol.
2014;51(6):721–729.
[49] Rossol M, Meusch U, Pierer M, et al. Interaction between trans-membrane TNF and TNFR1/2 mediates the activation of monocytes by contact with T cells. J Immunol.2007;179(6):4239– 4248.
[50] Tavakkoli F, Nahavandi M, Wyche MQ, Perlin E. Plasma levels of TNF-alpha in sickle cell patients receiving hydroxyurea.Hematology.
2004;9(1):61–64.
[51] Sarray S, Saleh LR, Lisa Saldanha F, Al-Habboubi HH, Mahdi N, Almawi WY. Serum IL-6, IL-10, and TNFalpha levels in pediatric sickle cell disease patients during vasoocclusive crisis and steady state condition.Cytokine.2015;72(1):43–47.
TNF-alpha blockers in rheumatoid arthritis: a meta-analysis. Mod Rheumatol.2013;23(3):489–495.
[53] Hoppe C, Klitz W, D’harlingue K, et al. Confirmation of an association between the TNF(-308) promoter polymorphism and stroke risk in children with sickle cell anemia.Stroke.2007;38(8):2241–2246.
[54] Chang Milbauer L, Wei P, Enenstein J, et al. Genetic endothelial systems biology of sickle stroke risk. Blood. 2008;111(7):3872– 3879.
[55] Yang WS, Han NJ, Kim JJ, Lee MJ, Park SK. TNF-a activates high-mobility group Box 1 - toll-like receptor 4 signaling path-way in human aortic endothelial cells. Cell Physiol Biochem.
2016;38(6):2139–2151.
S U P P O R T I N G I N F O R M A T I O N
Additional Supporting Information may be found online in the
sup-porting information tab for this article.
How to cite this article:Solovey A, Somani A, Belcher JD, et al.
A monocyte-TNF-endothelial activation axis in sickle transgenic
mice: Therapeutic benefit from TNF blockade.Am J Hematol.
2017;92:1119–1130.https://doi.org/10.1002/ajh.24856
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